KR100619546B1 - 2 / 2 Human Monoclonal Autoantibodies to Histones H2A and/or H2B and the Segment Thereof - Google Patents

Abstract

The present invention relates to human monoclonal autoantibodies or fragments thereof against histone H2A and / or H2V, more specifically histone H2A and / or isolated from peripheral blood leukocytes (PBL) of SLE patients using phage display. A human antihistone mAb specific for H2B and fragments thereof.

The antibody or fragment thereof according to the present invention contains a heavy chain variable region and a light chain variable region specific for histones H2A and H2B derived from PBL of an SLE patient, and thus is useful for the treatment of autoimmune diseases including human SLE.

Anti Histone, SLE, Phage Display, Autoantibody, H2A, H2B

Description

Human Monoclonal Autoantibodies to Histones H2A and / or H2B and the Segment Thereof}             

Figure 1 shows the amino acid sequence of the heavy chain variable region of the anti histone monoclonal antibody (mAbs). Residues involved in the R mutations are shown in italics, and residues identical to the germline sequence are shown in dashes. EMBL / GenBank accession numbers are as follows: SLEhis 16: AY569442; SLEhis 18: AY569444; SLEhis 19: AY569446; And SLEhis 22: AY569448.

Figure 2 depicts the amino acid sequence of the light chain variable region of anti histone mAbs. Residues involved in the R mutations are shown in italics and residues identical to the germline sequence are shown in dashes. EMBL / GenBank accession numbers are as follows: SLEhis 16: AY569449; SLEhis 18: AY569443; SLEhis 19: AY569445; And SLEhis 22: AY569447.

Figure 3 shows immunoblotting analysis of soluble Fabs of SLEhis22 according to the present invention. Histones H1 (lane 1), H2A (lane 2), H2B (lane 3), H3 (lane 4), and H4 (lane 5) were separated by SDS-PAGE. The resulting histones were stained with Coomassie bluant blue (A), and analyzed by immunoblotting using soluble Fab from SLEhis22 (B).

Figure 4 shows the ELISA results for soluble Fab of SLEhis22 according to the present invention.

Field of invention

The present invention relates to human monoclonal autoantibodies or fragments thereof against histone H2A and / or H2V, more specifically histone H2A and / or isolated from peripheral blood leukocytes (PBL) of SLE patients using phage display. A human antihistone mAb specific for H2B and fragments thereof.

Background of the Invention

Histones are major autoantigens in autoimmune diseases such as some systemic lupus erythematosus (SLE). Antihistone antibodies (Abs) are detected in the serum of mice such as NZB / NZW and MRL / Mp-1pr / lpr strains that are susceptible to SLE patients and naturally occurring autoimmune diseases (Gioud et al., J. Immunol., 131: 269, 1983; Monestier et al., Clin.Exp. Immunol., 99:37, 1995; Brick et al., Clin. Immunol. Immunopathol., 54: 372, 1990), type 1 It is detected in patients with autoimmune infections (Chen et al., J. Gastroenterol. Hepatol., 13, 483, 1998). Production of such autoantibodies is correlated with disease activity (Gioud et al., J. Immunol., 131: 269, 1983; Schett et al., Arthritis Rheum., 43: 420, 2000; O ' Dell et al., Clin. Immunol. Immunopathol., 47: 343, 1988; Teodorescu et al., Clin. Immunol., 110: 145, 2004).

Specific molecular analysis of autoantibodies is important for understanding the basic mechanisms of autoantibody production and their role in pathogenesis. The structure and properties of some antihistone mAbs have only been reported in mice (Monestier et al., Mol. Immunol., 30: 1069, 1993; Di Valerio et al., J. Immunol., 155: 2258, 1995; Monestier, Eur. J. Immunol., 21: 1725, 1991). Although it has been reported that mAbs with different specificities for different histones and H2A and H2B histone fragments have been isolated from lupus mice, information on the variable (V) region of human antihistone Ab is not yet known.

Therefore, the present inventors isolated four human antihistone mAbs specific for histones H2A and H2B from peripheral blood leukocytes (PBL) of SLE patients using phage display, and determined the nucleotide and amino acid sequences of the heavy and light chain variable regions. The present invention was completed by confirming that the antibody or fragment thereof can be prepared for analysis by applying to SLE patients.                         

It is therefore an object of the present invention to provide antibodies or fragments thereof for application to SLE patients, which contain heavy and light chain variable regions specific for histones H2A and / or H2B derived from PBLs of SLE patients.

In order to achieve the above object, the present invention provides an antibody or a fragment thereof having the amino acid sequence of SEQ ID NO: 1 as a light chain variable region and having the amino acid sequence of SEQ ID NO: 2 as a heavy chain variable region. In the present invention, the antibody or fragment thereof may be characterized by specifically binding to histone H2A and / or H2B.

The present invention also provides a DNA sequence encoding the antibody or fragment thereof. In the present invention, the DNA sequence encoding the light chain and heavy chain variable region may be characterized in that SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

The present invention also provides a light chain variable region of the antibody consisting of the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region of the antibody consisting of the amino acid sequence of SEQ ID NO: 2, respectively.

The invention also relates to the heavy chain CDRs of histone H2A and / or H2B specific antibodies selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 and in the group consisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 Each provides a light chain CDR of a histone H2A and / or H2B specific antibody of choice.

The present invention also provides an antibody or fragment thereof having an amino acid sequence of SEQ ID NO: 1 as a light chain variable region and an antibody or fragment thereof having an amino acid sequence of SEQ ID NO: 2 as a heavy chain variable region, respectively.

The present invention also provides an antibody or fragment thereof having the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 as heavy chain CDRs.

The present invention also provides an antibody or fragment thereof having the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 as light chain CDRs, respectively.

The present invention also provides an antibody or fragment thereof having the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 as heavy chain CDRs, and having the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 as light chain CDRs do.

The present invention also provides a human antibody or fragment thereof prepared using the antibody or fragment thereof. In the present invention, the human antibody or fragment thereof may be prepared by a phage display method.

Histones are cationic proteins that bind to DNA to form nucleosomes. Serum in patients with SLE contains individual histones, more complex determinants (eg, H2A-H2B dimers), or autoantibodies to histone peptides (Monestier et al., Mol. Immunol , 30: 1069, 1993; Monestier et al., Rheu. Dis. Clin.N. Am., 18: 415, 1992; Stemmer et al., Clin. Immunol.Immunopathol., 76:82, 1995).

To determine the structural characteristics of human antihistone autoantibodies, we analyzed four mAb variable regions cloned from peripheral blood lymphocytes (PBLs) of systemic lupus erythematosus (SLE) patients. SLEhis22, a monoclonal autoantibody against human histones H2A and H2B, was specific for histones H2A and H2B.

The sequence numbers cited in the present invention are as follows.

SEQ ID NO: Amino acid sequence of the SLEhis22 light chain variable region

SEQ ID NO: 2 amino acid sequence of the SLEhis22 heavy chain variable region

SEQ ID NO: 3: DNA sequence encoding amino acid of SLEhis22 light chain variable region

SEQ ID NO: 4: DNA sequence encoding amino acid of SLEhis22 heavy chain variable region

SEQ ID NO: 5 amino acid sequence of SLEhis22 CDRH1

SEQ ID NO: 6 amino acid sequence of SLEhis22 CDRH2

SEQ ID NO: Amino acid sequence of SLEhis22 CDRH3

SEQ ID NO: Amino acid sequence of SLEhis22 CDRL1

SEQ ID NO: Amino acid sequence of SLEhis22 CDRL2

SEQ ID NO: 10 amino acid sequence of SLEhis22 CDRL3

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Isolation of H2A and H2B Specific Antibodies Using Phage Display

(1) PCR amplification and library construction of antigen-binding fragment (Fab)

Total RNA was isolated from peripheral blood leukocytes (PBLs) of patients diagnosed with SLE who did not take any medication for SLE treatment (Ajou University Medical Center). The patient is the patient with the highest titer of antihistone Ab in screened serum from 10 patients. The single-strand cDNA was synthesized using the single-strand cDNA synthesis kit (Pharmacia) from the isolated total RNA, and then a human antigen-binding fragment (Fab) library was constructed using a three-step PCR series. In the primary PCR, individual V _H , V _L , C _H 1, and CL genes were amplified, respectively, and Fd and light chain sequences were generated via second overlapping extension PCR. Third-order overlapping extension PCR generated full length Fabs of Fd and Vκ or Fd and V _λ .

As a phagemid vector, pComb3X (GenBank Accession No. AF268281) contains two ribosomal-binding sites and a signal peptide. Individual Fds and light chains migrate to the periplasm and are assembled there and displayed on the surface of the phage particles. Each heavy chain variable and light chain variable 5 'primers for the gene family and 3' constant region primers for the IgG H chain and the κ or λ light chain were synthesized according to the methods described in the prior art (Barbas et al., A Laboratory Manual, 1st ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). There are asymmetric Sfi I restriction endonuclease sites on the 5 'and 3' ends of phage display vectors used to clone full length Fab PCR products (Andris-Widhopf et al. , J. Immunol. Meth. , 242: 159, 2000).

Approximately 1500 bp of final product was purified and then inserted into the pComb3X vector. XL-1 / Blue (Stratagene) competent cells were transformed with recombinant pComb3X containing Fab inserts. When XL-1 / Blue cells containing phagemids were infected with VCSM13 helper phage (Stratagene), production of packaged phage with DNA encoding Fab was possible.

(2) Selection of specific Fab clones and characterization of isolated clones

Selection of clones that specifically bind to histones (Roche) was performed according to the prior art (Barbas et al., 2001; Andris-Widhopf et al., 2000). Briefly, microtiter plates (Costar) were coated overnight at about 4 ° C. with 5 μg / ml histone at 0.1 M NaHCO ₃ , pH 7.6. After washing the plate, it was blocked for 2 hours at room temperature with 5% bovine serum albumin (BSA) dissolved in TBS (Tris-buffered saline; 10 mM Tris-HCl, 150 mM NaCl). After washing the plate once with TBS, 40 μl of freshly prepared phage particles, 5 μl of 10% BSA, and 5 μl of 10 × TBS were added, then the mixture was incubated at 37 ° C. for two hours.

The wells were then washed five times with TBS containing 0.5% Tween 20 (TBST) and incubated twice at room temperature with 50 μl of 0.1 M HCl / glycine (pH 2.2) to separate the combined phage particles. . The separated particles were neutralized by the addition of 15 μl of 1M Tris / HCl (pH 8.0), followed by the addition of 2 ml of actively growing XL-1 / Blue cells (Stratagene) to the neutralized 115 μl of phage particles. The mixture was left at room temperature for 15 minutes.

Next, SB (Super Broth; 30 g tryptone, 20 g yeast extract, 10 g Mops / l, pH 7) containing 50 µg / ml of 6 ml carbenicillin and 20 µg / ml of tetracycline was added. Cells were added and cells were added to 1 ml of VCSM13 and then grown at 37 ° C. for two hours. SB containing carbenicillin / tetracycline was added in a 1 L flask to a final volume of 100 ml. After two hours at 37 ° C., kanamycin (50 μg / ml) was added and the cultures were grown overnight at 37 ° C. After 15 minutes of growth, the cultures were separated by centrifugation at 3000 g at 4 ° C. To the supernatant was added PEG-8000 (4%) and NaCl (3%) and the mixture was incubated on ice for 30 minutes.

Next, for 15 minutes, centrifuged at 15,000 g at 4 ° C., then the supernatant was removed and the phage pellet was resuspended in 2 ml of 1% BSA / TBS. The suspended solution was used for the second round panning. After the second round of panning, phage supernatants and phagemid DNA were recovered and the supernatants used for phage ELISA.

(3) phage ELISA

Phage supernatants were selected based on antibody binding specificity for the antigen. Microtiter plates were coated with 5 μg / ml histone dissolved in 0.1 M NaHCO ₃ (pH 7.6) overnight at 4 ° C. After blocking with 5% BSA / TBS, phage supernatants were added in duplicate to the wells and plates were incubated at 37 ° C. for two hours. Plates were then washed six times with TBST and reacted with horseradish peroxidase (HRP) -binding anti-M13 antibody (Amersham Pharmacia Biotech) diluted in 5% BSA / TBS for 1 hour at 37 ° C. After washing the plate six times with TBST, it was reacted with 50 μl of tetramethyl benzidine (TMB) substrate (ZYMED) and then stopped with 50 μl of 1M HCl. Finally, the absorbance at 450 nm was measured using an ELISA reader ELX 808 IU (BIO-TEK).

(4) Sequencing Fab Variable Region Genes

Phagemid DNA from positive clones was digested with Sfi I restriction enzymes in phage ELISA to analyze the inserted DNA size and sequenced using an automated sequencer (Biocore Technology). Sequence data were analyzed using BLAST (Basic Local Alignment Search Tool) and DNAPLOT (V BASE). Primers for the heavy and light chain variable regions are as follows:

SEQ ID NO: 11 (heavy chain variable region): 5'-ACCTATTGCCTACGGCAGCCG-3 '

SEQ ID NO: 12 (Light chain variable region): 5'-AAGACAGCTATCGCGATTGCAG-3 '

(5) Preparation of Soluble Fab

To produce soluble Fabs that do not contain phage coat protein pIII, electrocompetent TOP10F 'cells (Invitrogen), a non-suppressive cell line that recognizes amber codons as stop codons, are transformed with phagemids containing phage Fabs I was. Individual colonies were incubated at 37 ° C. in SB medium containing carbenicillin, tetracycline and 20 mM MgCl ₂ until the absorbance at 600 nm was between 0.4 and 0.5.

Fab expression was induced by incubating the cells overnight at 30 ° C. in the presence of 1 mM isopropyl thiogalactopyranoside. After the end of the culture the cells were centrifuged and the cell pellet was resuspended in 10 ml ice-cold TES buffer (0.2 M Tris-HCl, pH 8.0, 0.5 mM EDTA, 0.5 M sucrose) per liter culture. Next, ice-cold TES buffer diluted 1: 3 with distilled water was added and the mixture was incubated for 30 minutes on ice. The mixture was then centrifuged at 15,000 g for 20 minutes and the supernatant was used as a soluble periplasm extract. Soluble Fabs were also obtained from expression induced cell culture supernatants.

(6) Immunoblot Analysis of Soluble Fabs

Soluble Fab was separated by 15% SDS-PAGE and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was blocked with 5% Scheme Milk / TBST for 1 hour at room temperature and then washed with TBST and then anti-human IgG (Fab specific) mAb-HRP diluted 1: 1000 in 1% Scheme Milk / TBST ( Sigma) was reacted at room temperature for 1 hour. The membrane was then further washed with TBST and immunoreactive protein was detected with ECL reagent (Amersham Pharmacia).

For immunoblotting analysis of soluble Fabs that bind histones, individual histones (Roche) were separated by 15% SDS-PAGE. For Fab reactivity analysis, histones isolated on PVDF membranes were incubated with soluble Fabs.

(7) direct binding ELISA for histone binding

Direct binding ELISA was performed in the same manner as described for phage ELISA except that the soluble Fabs were incubated in histone coated microtiter wells instead of phage supernatants. Bound Fab was reacted with biotin binding anti-human goat IgG (Sigma) and streptavidin-alkaline phosphatase (Sigma) diluted 1: 40,000 in 1% BSA / TBS. Alkaline phosphatase substrate was added to each well, and then the absorbance at 405 nm was read with ELISA reader ELX 808 IU (BIO-TEK).

Example 2: Sequence Analysis of Human Antihistone Antibodies

To analyze the specific molecular structure of human antihistone mAb, cDNA generated from RNA purified from PBL of SLE patients was used. First, four mAbs specific for histone H2A and H2B (SLEhis16; SLEhis18; SLEhis19; and SLEhis22) were generated by phage display, and the light chain variable region and heavy chain variable region DNA sequences were determined. SLEhis22, the most efficient of the antigen binding, had the DNA sequences of the light chain variable region and the heavy chain variable region of SEQ ID NO: 3 and SEQ ID NO: 4, respectively. SLEhis22 uses the V _H 3 gene family, which is the largest gene family in humans, and is derived from the A27 member of the V _K III gene family (see Table 1).

Fab clone VH gene usuage                                              CDR3-VH length (a.a) IP of VH VL gene usage                                              CDR3-VL length (a.a) IP of VL VH family DH JH VL family JL SLEhis16 4-59 D3-3 JH6 17 8.6 O8 JK3 9 5.1 SLEhis18 5-51 D6-19 JH4 11 6.1 L25 JK2 9 8.7 SLEhis19 3-33 D4-23 JH1 12 9.0 L6 JK1 8 9.1 SLEhis22 3-33 Unknown JH5 10 9.3 A27 JK1 9 8.7

Example 3: Characteristics of Antibodies or Fragments thereof

(1) primary structure analysis

Compared to the germline counterparts of the gene fragments used by the mAb, the SLEhis22 heavy chain variable region is a CDR, unlike other mAbs that have a high rate of mutation in both CDRs or framework regions. No substitution mutation or silent mutation was found in E. coli, and only four substitution mutations were found to contain only FR (Table 2).

No. of clones (isotypes) Identity to GL (%) No. of mutation                                              R / S ratio                                              FR1 CDR1 FR2 CDR2 FR3 CDR ’S FR ’S R S R S R S R S R S SLEhis16 VH 96 2 0 0 0 One One One One One 4 1/1 4/5 SLEhis18 VH 91 One 3 0 0 0 0 7 2 5 4 7/2 6/7 SLEhis19 VH 96 One 0 0 One One 0 One One 3 One 1/2 5/1 SLEhis22 VH 98 0 0 0 0 0 0 0 0 4 0 0/0 4/0 SLEhis16 VL 97 0 One 0 0 One One 2 0 One 0 2/0 2/2 SLEhis18 VL 92 3 One 3 2 0 3 0 0 3 One 3/2 6/5 SLEhis19 VL 95 0 0 4 0 0 0 One 0 2 3 5/0 2/3 SLEhis22 VL 97 0 One One 0 0 0 0 0 One One 1/0 1/2

The similarity between the SLEhis22 mAb heavy chain variable region gene and the dot line genes was 98% higher than that of other mAbs, and similarity with the point line gene in the light chain variable region gene was 97%. The overall R / S ratios were 0/0 and 1/0 in the CDRs of the SLEhis22 mAb heavy and variable chain genes, and the overall R / S ratios were 4/0 and 1 in the FRs of the heavy and variable chain genes. Was / 2.

As a result of sequencing of the antibody, it was confirmed that the heavy and light chain variable regions include many negatively charged amino acid residues (aspartate and glutamate) throughout (FIG. 1 and FIG. 2). 1 shows the amino acid sequence of the heavy chain variable region of anti-histone mAbs. Residues involved in the R mutations are shown in italics, and residues identical to the germline sequence are shown in dashes. EMBL / GenBank accession numbers are as follows: SLEhis 16: AY569442; SLEhis 18: AY569444; SLEhis 19: AY569446; And SLEhis 22: AY569448. Figure 2 depicts the amino acid sequence of the light chain variable region of anti histone mAbs. Residues involved in the R mutations are shown in italics, and residues identical to the germline sequence are shown in dashes. EMBL / GenBank accession numbers are as follows: SLEhis 16: AY569449; SLEhis 18: AY569443; SLEhis 19: AY569445; And SLEhis 22: AY569447. Most of CDR2 and CDR3 of the heavy chain variable region comprise one or more negatively charged amino acid residues.

(2) Determination of binding properties through immunoblotting

To confirm the binding properties of mAbs, TOP10F 'cells were transformed with phagemid containing phage Fab. After inducing gene expression in the transformed cells, soluble Fabs were obtained from extracts of the culture supernatant or periplasm.

Next, the binding properties of Fab to individual histones were analyzed by immunoblotting. The identified molecular weight of Fab was about 27 kDa. 3 shows immunoblotting analysis of soluble Fabs that bind histones. Histones H1 (lane 1), H2A (lane 2), H2B (lane 3), H3 (lane 4), and H4 (lane 5) were separated by SDS-PAGE. (A) is a photograph of the resulting histone stained with Coomassie bluant blue, (B) is a photograph (B) is analyzed by immunoblotting using soluble Fab from SLEhis22.

As shown in FIG. 3A, in the lanes containing histones H2A, H2B, and H3, there was a major band of 15-20 kDa, and the unidentified minor band was at a high molecular weight position. Histone H1 was detected as the main band at a molecular weight position slightly larger than 30 kDa, whereas the major band of histone H4 was 15 kDa. The reactivity of the soluble Fabs with the individual histones is shown in Figure 3B. SLEhis22 reacted strongly with H2A, H2B and H3 and very weakly with H1 or H4. The same pattern but overall weak reactivity was obtained with other mAbs.

(3) Determination of binding properties through ELISA

In order to further investigate the binding characteristics of the SLEhis22 antibody according to the present invention, a direct binding ELISA using an individual histone as an antigen coated on a microtiter plate was performed. Microtiter plate wells were coated with each histone or mixed histones and incubated with soluble Fabs obtained from each mAb. As a result, as shown in Figure 4, anti-histone mAb (SLEhis22) according to the present invention showed a relatively weak binding to H3, showed the strongest binding to histone H2A, and then showed a strong binding to H2B. While it did not bind to histone H1, H4, or double-stranded DNA. The binding of antihistone mAb to H3 or total histones according to the present invention was weak compared to binding to H2A and H2B. From this result, it was confirmed that the antihistone mAb (SLEhis22) according to the present invention binds specifically to H2A and / or H2B.

As described in detail above, the present invention has the effect of providing antihistone human monoclonal autoantibodies and fragments thereof specific for histone H2A and / or H2VIII. Antibodies and fragments thereof according to the present invention contain heavy chain and light chain variable regions specific for histones H2A and H2B derived from PBLs of SLE patients and are expected to be useful for the treatment of autoimmune diseases including human SLE.                     

As described above in detail the specific parts of the present invention, for those of ordinary skill in the art, these specific descriptions are only preferred embodiments, which are not intended to limit the scope of the present invention. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

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Claims (15)

An antibody or antigen-binding fragment thereof (Fab) having the amino acid sequence of SEQ ID NO: 1 as the light chain variable region and the amino acid sequence of SEQ ID NO: 2 as the heavy chain variable region. The antibody or antigen-binding fragment thereof (Fab) according to claim 1, which binds specifically to histone H2A and / or H2B. A DNA sequence encoding the antibody of claim 1 or 2 or an antigen-binding fragment thereof. 4. The DNA sequence of claim 3, wherein the DNA sequences encoding the light and heavy chain variable regions are SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The light chain variable region of the antibody consisting of the amino acid sequence of SEQ ID NO: 1. Heavy chain variable region of the antibody consisting of the amino acid sequence of SEQ ID NO: 2. A heavy chain CDR of a histone H2A and / or H2B specific antibody selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7. Light chain CDRs of histone H2A and / or H2B specific antibodies selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10; delete delete delete delete An antibody or antigen-binding fragment thereof (Fab) having the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 as the heavy chain CDR, and having the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 as the light chain CDRs. delete delete

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